Tissue implant systems

ABSTRACT

An implant assembly for a host tissue is provided. The implant assembly comprises a pouch including wall means defining a chamber for holding a second member, the wall means including an outer vascularizing membrane having a conformation that results in growth of vascular structures by the host tissue close to an interface between the vascularizing membrane and host tissue. The assembly includes a second member that can be removably inserted in the chamber including an interior for receiving cells and wall means defining an immuno-isolating membrane that isolates the cells from the immune response of the host tissue.

RELATED APPLICATION

This application is a continuation-in-part of copending U.S. Ser. No.08/210,068, filed Mar. 17, 1994, still pending which is a continuationof U.S. Ser. No. 07/933,871 entitled: "Close Vascularization ImplantMaterial" filed Aug. 21, 1992 abandoned, which is a continuation-in-partof U.S. Ser. No. 07/735,401 entitled: "Close Vascularization ImplantMaterial" filed Jul. 24, 1991 abandoned, which is a continuation-in-partof U.S. Ser. No. 07/606,791 entitled: "Membrane for Vascularization ofForeign Body Capsule" filed Oct. 30, 1990 abandoned.

FIELD OF THE INVENTION

The inventions relate to systems and methods for implanting living cellswithin a host.

BACKGROUND OF THE INVENTIONS

For several years, researchers have been trying to surgically implantliving cells in a host to treat various cell and molecular deficiencydiseases. In theory, the implanted cells will generate biologicalproducts that the host, because of disease or injury, cannot produce foritself. For example, the implant assembly can contain pancreatic cells(clusters of which are called "islets"), which generate insulin that adiabetic host lacks.

Yet, in practice, conventional implant assemblies and methodologiesusually fail to keep the implanted cells alive long enough to providethe intended therapeutic benefit. For example, pancreatic cellsimplanted for the treatment of diabetes usually die or becomedysfunctional within a few days or weeks after implantation.

For a period after implantation, the region of the host tissue next tothe implant assembly can be characterized as ischemic. "Ischemic" meansthat there is not a sufficient flow of blood in the tissue regionclosely surrounding the implant assembly. Usually, this ischemiccondition exists during the first two weeks of implantation. Mostimplanted cells fail to live through this period.

During the ischemic period, a foreign body capsule forms around theimplanted cells. The capsule consists of flattened macrophages, foreignbody giant cells, and fibroblasts. Conventional hypotheses blame theforeign body capsule for causing implanted cells to die or becomedysfunctional during the ischemic period.

The inventors have discovered that these widely held hypotheses arewrong. The inventors have discovered that the cells do not die becauseconventional implant assemblies and methodologies themselves lack theinnate capacity to support the implanted cells' ongoing life processesduring the critical ischemic period, when the host's vascular structuresare not nearby. Because of this, the implanted cells perish before thehost can grow new vascular structures close enough to sustain them.

When implanted cells die during the ischemic period, a classical foreignbody capsule inevitably forms around the implant. The persistentpresence of this capsule led previous researchers to the falseconclusion that the host's foreign body reaction was the cause ofimplanted cell death, rather than its result.

The invention corrects these and other problems in existing implantassemblies and methodologies.

Many previous implant assemblies have also failed to be useful in aclinical setting, because they cannot be practically implanted andtolerated by the host without danger or discomfort.

For example, an implant assembly that housed cells within hollow fiberswas used by CytoTherapeutics to successfully treat diabetes in rats. Theassembly consisted of 7 fibers, each being 2 cm long and 0.073 cm indiameter. The pancreatic cells were present within the fibers at adensity of about 25,000 cells per cm³. For this assembly to beclinically useful for the treatment of diabetes in humans, it would haveto contain at least about 250,000 pancreatic islets (each islet containsabout 1000 cells). This means that, to hold enough pancreatic cells totreat human diabetes, the assembly would have to be about 117 feet long.This makes the assembly unusable for clinical use in humans.

Cells have also been encapsulated in tiny hydrogel vessels, calledmicrocapsules. These tiny vessels cannot be implanted within the host'ssoft tissues, because they lack the physical strength to withstand thephysiological stresses normally encountered close to the host tissue.Instead, the microcapsules are suspended in a free floating state withina solution that is infused into the host's peritoneal cavity.

In reality, the microcapsules have only limited clinical application.Not all persons can tolerate their injection free of danger ordiscomfort. Microcapsules are non-adhesive, and they do not stick toorgans. Instead, they settle in large masses at the bottom of theperitoneal cavity. And, if implanted directly within the host's tissue,the microcapsules will rupture, and the contained cells would perish.For these reasons, microcapsules fail to provide a widely usableclinical solution to the problems surrounding the therapeuticimplantation of cells.

Once implanted, a potential disadvantage of the known system is thatonce the cells die or are no longer sufficiently viable, a surgeon mustimplant a new device.

Additionally, many known devices seal the cells between layers ofplastic. It is not possible to test the devices to determine theintegrity of the system.

SUMMARY OF THE INVENTION

The present invention provides an improved implantable device. Theimplantable device can contain living cells capable of chemicalinteraction with host tissue.

To this end, in an embodiment, an implant assembly is provided for ahost tissue comprising a pouch including wall means defining a chamberfor holding a second member, the wall means including an outervascularizing membrane having a conformation that results in growth ofvascular structures by the host tissue close to an interface between thevascularizing membrane and host tissue. The assembly includes a secondmember that can be removably inserted in the chamber including aninterior for receiving cells and wall means defining an immuno-isolatingmembrane that isolates the cells from the immune response of the hosttissue.

In an embodiment, the pouch includes a resealable opening.

In an embodiment, the wall means define a chamber that has asubstantially rectangular cross-sectional shape.

In an embodiment, the assembly includes means for resealing an openingof the pouch. The means for resealing the opening includes a removablemechanical means for sealing an opening at an end of the pouch member.

In an embodiment, the second member includes a spacer that defines, inpart, the interior for receiving cells.

In an embodiment, the wall means of the pouch defines a structure havinga substantially pillow-like shape.

In an embodiment, the assembly includes means for securing the secondmember in the pouch.

In another embodiment, the present invention provides an implantassembly for a host tissue comprising a first member including wallmeans that define a chamber for holding a second member, the wall meansincluding a membrane defining an interface with the host tissue, themembrane having a conformation that results in growth of vascularstructures by the host tissue close to the interface. The assemblyincludes a second member that is removably received in the first memberand including a cavity having cells and a membrane having a structurethat isolates cells within the cavity from the immune response of thehost.

In still another embodiment, a method for implanting cells into a hosttissue is provided comprising the steps of: providing a pouch includingwall means having an outer vascularizing membrane having a conformationthat results in growth of vascular structures by the host tissue closeto an interface between the vascularizing membrane and host tissue;adding cells to an interior of a cassette that includes animmuno-isolating membrane that isolates the implanted cells from theimmune response of host tissue; inserting the cassette into the implantassembly through an opening; and sealing the opening.

In an embodiment of the method, the pouch is implanted into a hostbefore the cassette is added thereto.

In an embodiment of the method, the cassette is added to the pouch aftervascularization of the pouch.

In an embodiment of the method, the cassette is tested before beinginserted into the pouch.

In an embodiment of the method, the pouch is sealed by a clip.

In an embodiment of the method, the pouch is sealed by a heat seal.

In an embodiment, the method includes the steps of: opening the pouch;removing the cassette; and adding to the pouch a different cassette.

An advantage of the present invention is that it provides a device forimplanting cells that allows the implanted cells to be replaced.

Another advantage of the present invention is that it is capable of anon demand drug delivery offering an alternative to bolus or IV drugadministration.

Further, an advantage of the present invention is that it offerspotential treatment therapies including internal delivery of insulin,factor VIII, factor IX, etc.

Another advantage of the present invention is that it provides a devicethat may be implanted using less invasive surgical techniques.

Still further, an advantage of the present invention is that thevascularizing membrane can be implanted separate from theimmuno-suppressive cell containing device.

Still further, an advantage of the present invention is that it providesa device that can be replaced or removed with minimal local vasculardamage.

Still further, an advantage of the present invention is that the devicecan be filled and tested prior to implantation.

Additional features and advantages of the present invention aredescribed in, and will be apparent from, the detailed description of thepresently preferred embodiments and from the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an implant assembly that embodies thefeatures of the invention being held in the hand of a practitioner;

FIG. 2 is an enlarged perspective view of the implant assembly shown inFIG. 1;

FIG. 3 is an enlarged and exploded perspective view of the implantassembly shown in FIG. 2;

FIG. 4 is a side section view of the implant assembly taken generallyalong line 4--4 in FIG. 2;

FIG. 5 is an enlarged and exploded perspective view of another implantassembly that embodies the features of the invention, showing thepractitioner loading implanted cells into the assembly;

FIG. 6 is an enlarged assembled view of the assembly shown in FIG. 5,before the formation of a peripheral seal;

FIG. 7 is an enlarged view of the assembly shown in FIG. 6, partiallypeeled apart to show the interior;

FIG. 8 is an enlarged assembled view of the assembly shown in FIG. 5after the formation of a peripheral seal;

FIG. 9 is a side section view of a portion of the sealed assembly takengenerally along line 9--9 in FIG. 8;

FIG. 10 is a side section view of the assembly before sealing, takengenerally along line 10--10 in FIG. 6;

FIG. 11 is a perspective view of a lamination slide holding the bottomlayer of the laminated boundary structure that embodies the features ofthe invention;

FIG. 12 is a side section view of the lamination slide taken generallyalong line 12--12 in FIG. 11;

FIG. 13 is a perspective view of several lamination slides laid side byside for the application of adhesive filaments in the process of makingthe laminated boundary structure;

FIG. 14 is a side section view of the laminated boundary structure withits top layer laid over the cement filaments applied in FIG. 13;

FIG. 15 is a side section view of the laminated boundary structureclamped between two lamination slides while the cement filaments cure;

FIG. 16 is a perspective view of individual boundary wall elements beingcut from the laminated structures made following the steps shown inFIGS. 11 to 15;

FIG. 17 is a diagrammatic depiction of an implant assembly that embodiesthe features of the invention after having been surgically implanted inhost tissue;

FIG. 18 is a diagrammatic depiction of an embodiment of the implantassembly during the ischemic period, after about one or two days ofimplantation, showing the surrounding wound area filled with exudate;

FIG. 19 is a diagrammatic depiction of the implant assembly after abouttwo weeks of implantation, showing the formation of vascular structuresclose to the boundary, ending the ischemic period;

FIG. 20 is a diagrammatic depiction of a section of the implant assemblyin which the implanted cells have survived the ischemic period, showingthe formation of vascular structures close to the boundary and theresulting alteration of the foreign body capsule;

FIG. 21 is a diagrammatic depiction of a section of the implant assemblyin which the implanted cells have not survived the ischemic period,showing the lack of vascular structures close to the boundary and theresulting intervention of the foreign body capsule;

FIG. 22 is a graph showing the therapeutic loading curve for pancreaticcells derived in accordance with the invention.

FIG. 23 illustrates schematically another embodiment of an implantassembly of the present invention.

FIG. 24 is a micrograph of a membrane material;

FIG. 25 is a micrograph of the membrane material of FIG. 24 at adifferent magnification;

FIG. 26 is a micrograph of the membrane material of FIG. 24 at adifferent magnification;

FIG. 27 is a micrograph of another membrane material;

FIG. 28 is a micrograph of the membrane material of FIG. 27 at adifferent magnification; and

FIG. 29 is a micrograph of still another membrane material.

Before explaining the preferred embodiments, it is to be understood thatthe inventions are not limited in use to the details of construction ormethodologies there set forth or as illustrated in the drawings. Theinventions are capable of other embodiments and of being practiced andcarried out in various ways.

DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

FIGS. 1 to 4 show an implant assembly 10 that embodies features of theinvention.

The assembly 10 can carry preselected types of living cells 12 forimplanting within the soft tissue of a host. The implanted cells 12generate biological products that the host, because of disease orinjury, cannot produce for itself.

For example, the implant assembly 10 can carry clusters of pancreaticcells (called "islets"), which generate insulin for release into and useby a diabetic host.

The assembly 10 forms a porous, life sustaining boundary between theimplanted cells 12 and the host. The porous boundary isolates theimplanted cells 12 from attack and destruction by certain biologicalmechanisms of the host. At the same time, the porous boundary associateswith the host's biological system closely enough to transfer nutrientsand wastes in support of the biological processes of the implanted cells12. The porous boundary also transfers the therapeutic productsgenerated by the implanted cells 12 to the host.

In the embodiment shown in FIGS. 1 to 4, the assembly 10 includes ahoop-like housing 11. The housing 11 includes a first hoop element 14and a second hoop element 16 that nests within the first hoop element14. The assembly 10 also forms a cell chamber 18 within the hoop-likehousing 11.

The first hoop element 14 has an upstanding cylindrical side wall 20that peripherally defines an open area. First and second centralopenings 22 and 24 lead into the open area. The first central opening 22is smaller than the second central opening 24. This forms an interiorstep or ledge 26 next to the first opening 22.

The second hoop element 16 also has a central opening 28. The secondhoop element 16 has an outer diameter that is slightly greater than theinner diameter of the open area of the first hoop element 14. Theperipheral edge of the second central opening 16 contains a slightchamfer 30 to receive the second hoop element 16. When assembled, thesecond hoop element 16 nests snugly in an interference press fit withinthe open area of the first hoop element 14 (see FIG. 2).

The first hoop element 14 and the second hoop element 16 are made of adurable biocompatible ceramic or metallic material, like titanium. Liketitanium, the selected material should also preferably be subject todetection within the host tissue by fluoroscopy, x-ray, and the like.

The specific dimensions of the hoop-like housing 11 may vary accordingto its intended use and the volume of cells 12 it contains.

In one preferred embodiment, the side wall of the first hoop element 14is about 0.055 inch in height and has an outer diameter of about 0.375inch. The open area has an inner diameter of about 0.325 inch where itjoins the inner edge of the chamfer 30 of the second central opening 24.The second central opening 24 has an inner diameter of about 0.326 incharound the outer edge of the chamfer 30. The first central opening 14has an inner diameter of about 0.275 inch and a depth of about 0.015inch, where it joins the interior ledge 26.

In this embodiment, the associated second hoop element 16 has a heightof about 0.025 inch; an outer diameter of about 0.326; and an innerdiameter (for its central opening 28) of about 0.250 inch. The range ofinterference necessary to snugly join the second hoop element 16 withinthe first hoop element 14 will of course depend upon the nature of thematerials selected.

The chamber includes a first porous wall element 32, a second porouswall element 34, and a sealing gasket or ring 36 that is sandwichedbetween them. The sealing ring 36 is made of a mesh polyester material.

The wall elements 32 and 34 and sealing ring 36 are sized to fit snuglywithin the confines of the hoop-like housing 11. And, as will bedescribed in greater detail later, at least one (and preferably both)porous wall elements 32 and 34 have certain physical characteristicsselected to protect and sustain the viability of the cells 12 within thehost.

The ring 36 has a central open region 38. The open ring region 38,together with the overlying first and second porous wall elements 32 and34, create the chamber 18 to hold the implanted cells 12 (see FIG. 4).

In making the assembly 10 shown in FIGS. 1 to 4, the practitioner laysone wall element 32 onto the ledge 26 formed in the first hoop element14. The practitioner then lays the sealing ring 36 upon the wall element32. Next, the practitioner inserts the desired amount of cells 12 to beimplanted into the open region 38 of the ring 36. The amount ofimplanted cells 12 is sufficient to induce the expected therapeuticresponse in the host.

The practitioner next lays the other wall element 34 over the first wallelement 32, sealing ring 36, and inserted cells 12. To complete theassembly 10, the practitioner presses the second hoop element 16 throughthe second central opening 24 into pressing engagement against theadjacent wall element 34. This seals the periphery of the cell holdingchamber 18, which now snugly rests within the open area of the hoop-likehousing 11.

Once assembled, one wall element 32 rests against the interior ledge 26and is there exposed through the first central opening 22. The otherwall element 34 rests against the second hoop element 16 and is thereexposed through its central opening 28.

FIGS. 5 to 10 show another implant assembly 10' that embodies featuresof the invention. Like, the implant assembly 10 previously described,the assembly 10' includes a cell chamber 18' formed by first and secondporous wall elements 32' and 34' and an intermediate sealing ring 36'.

Unlike the first described implant assembly 10, the assembly 10' doesnot rely upon the hoop-like housing 11 to hold and seal the chamber 18'.Instead, a preformed peripheral weld 40 bonds and seals the edges of theporous wall elements 32' and 34' to the interior ring 36'.

In making the assembly 10' shown in FIGS. 5 to 10, the practitioner laysthe sealing ring 36' upon one wall element 32' and inserts the desiredamount of cells 12 to be implanted into the open region 38' of the ring36' (see FIG. 5). The practitioner overlays the other wall element 34'(as FIG. 6 shows). The practitioner then forms the weld 40 to seal theperipheral edges of the first and second wall elements 32' and 34' tothe ring 36' (as FIG. 8 shows). The weld compresses the peripheral edgeof the assembly 10' together, as FIG. 9 shows.

The practitioner selects a sealing technique that does not damage thecells 12 within the chamber 18'. For example, the inventors find thatsonic welding can be used without damage to the inserted tissue cells.

In a preferred embodiment (using the laminated structure 72 made asshown in FIGS. 11 to 16, as will be described later), the practitioneruses a Branson sonic welder. The welder is operated at 40 Khz, with941AES actuator, 947 m power supply, and 91 C power controller. The hornamplitude is about 1.4 mils and is operated at a hold time of about 0.3seconds; a weld time of about 0.20 seconds; a pressure of about 50 PSI;a trigger force of about 20 pounds; and a down speed of about 1.25(machine setting).

These are typical operating ranges for making the sonic weld and canvary according to the materials used and degree of cell loading withinthe chamber.

The integral assembly 10' formed in this manner can be implanteddirectly within host tissue, without use of an exterior housing.

Preferably, as FIG. 8 shows, the assembly 10' includes an attached clip42 made of a material that can be detected within the host tissue byfluoroscopy, x-ray, and the like. In this way, the practitioner caneasily locate the assembly 10' within the host, if required.

Like the first described embodiment, the specific dimensions of theassembly 10' may vary according to its intended use. And, like the firstdescribed embodiment, at least one (and preferably both) porous wallelements 32' and 34' have certain physical characteristics selected toprotect and sustain the viability of the cells within the host.

Regardless of the assembly used, the practitioner surgically implants itin the soft tissue 44 of the host (see FIG. 17). During surgery, thepractitioner positions the assembly 10 so that the exposed first andsecond wall elements 32 and 34 rest close to the surrounding host tissue44. In FIGS. 17 to 21, assembly 10 also encompasses assembly 10'.

The first and second wall elements 32 and 34 thereby together form thedesired boundary 46 between the biological system of the host tissue 44living outside the chamber 18 and the biological system of the implanttissue cells 12 living within the chamber 18.

For a period of time after implantation, the region of the host tissue44 immediately surrounding the implant assembly 10 is ischemic (see FIG.18). The region is ischemic, because the host treats the assembly 10 asa foreign body.

The host forms a wound area 48 around the assembly 10 (see FIG. 18). Thewound area 48 has spaces that become filled with wound exudate 50. Thewound exudate 50 keeps this area 48 ischemic.

Soon after implantation, host inflammatory cells enter and occupy theexudate area 48. "Inflammatory cells" include macrophages, foreign bodygiant cells, and fibroblasts.

The inflammatory cells try to remove the foreign implant assembly.Macrophages from the host try to ingest the foreign implant assembly 10.In some cases, the macrophages coalesce to form multinucleated giantcells. Fibroblast layers form to create a fibrous sac of cells andcollagen around the foreign implant assembly 10, commonly called theforeign body capsule 52 (see FIG. 20).

The inventors have discovered that it is not the foreign body capsule 52that most threatens the viability of the implanted cells during theischemic period. Rather, the existence of the cells is most threatenedduring the ischemic period when the boundary 46 itself fails to allowenough extracellular nutrients like glucose and other metabolic supportcompounds present at the boundary 46 to pass to the cells. Withoutmetabolic support, the implanted cells become dysfunctional or perish.

As FIG. 18 shows, the wound exudate 50 forms a fluid barrier between thevascular system of the host and the boundary 46. This barrier hindersthe extracellular passage of nutrients from the host vascular system tothe boundary 46. The concentrations of nutrients decrease as theytransit the exudate barrier to reach the boundary 46.

The host's inflammatory cells that in time enter the wound exudateregion 50 also create a metabolic sink. These cells compete for andfurther extract more of the host's extracellular nutrients before theyreach the boundary.

If the host is stimulated to grow new vascular structures 54 close tothe boundary 46, host endothelial cells will also enter the region 48.These cells begin the crucial process of forming the new vascularstructures 54. Still, their presence further contributes to themetabolic sink effect. The host's endothelial cells further reduce theavailability of nutrients for the implanted cells.

The ischemic period will end, if enough neovascular structures 54 fromthe host grow within the exudate region 50 close to the boundary 46 ofthe assembly 10 (as FIGS. 19 and 20 show). The close vascular structures54 shorten the extracellular path that nutrients must travel to reachthe boundary 46. The close vascular structures 54 provide nutrients inhigher concentrations to the implanted cells. Close vascularization alsotransports the therapeutic products generated by the implanted cells 12to the host.

However, all these desired benefits accrue only if the implanted cells12 survive the critical ischemic period.

The inventors have discovered that the diminished concentrations ofnutrients present at the boundary 46, although significantly reduced bythe exudate barrier and metabolic sink effects, are still enough tosustain the implanted cells. This is true, even in the presence of aforeign body capsule.

Still, the cells will die, if the boundary 46 itself lacks the capacityto let enough of the remaining nutrients through to the cells at asufficiently high rate. The inventors refer to this capacity as themetabolic transit value.

The inventors have discovered that the boundary 46 itself can alsopresent another significant barrier to the passage of nutrients. Theadded barrier effect of the boundary 46 can further reduce the alreadydiminished concentration of nutrients, until there is essentiallynothing left to sustain the cells.

The series barriers to the extracellular passage of nutrients (the woundexudate 50, the boundary 46, and the metabolic sink effect) also inhibitthe reverse passage metabolic wastes from the implanted cells.

The inventors have discovered that two principal factors threaten thesurvival of the implanted cells during the ischemic period. The firstfactor (which is conventionally recognized) is the failure to isolatethe cells from the natural immune response of the host. The secondfactor (which is not conventionally recognized) is the undesirableadditional barrier effect of the boundary 46 that impedes the essentialflux of already scarce nutrients to the implanted cells before closevascularization fully develops. The same barrier effect impedes the fluxof metabolic waste products away from the implanted cells to the host.

If the boundary 46 does not support the ongoing metabolic processes ofthe implanted cells while isolating them from the immune response of thehost during the ischemic period, the implanted cells will not live longenough to derive the benefits of close vascularization, if it occurs.

According to this aspect of the invention, then, the porous boundary 46is characterized in terms of its pore size; its ultimate physicalstrength; and its metabolic transit value. The first two characteristicsserve to isolate the implant tissue cells from the immune response ofthe host. The last characteristic serves to transfer nutrients and wasteproducts in support of the metabolic processes of implanted cells duringthe ischemic period, before close vascularization occurs. The lastcharacteristic sustains the viability of the implanted cells during theischemic period, even as a foreign body capsule forms.

According to another aspect of the invention, the assembly also includesan angiogenic material. The presence of an angiogenic materialstimulates the neovascularization required close to the boundary 46 tobring an end to the ischemic period.

According to yet another aspect of the invention, the porous boundary 46includes an interface 47 with the host tissue that is characterized by aconformation that supports and fosters the growth of vascular structuresby the host close to the boundary 46.

Further details of the beneficial characteristics of the boundary 46 andits associated host interface 47 will now be individually described.

Boundary Pore Size

The boundary 46 has a pore size sufficient to isolate the implant tissuecells from the immune response of the host.

As used in this Specification, "pore size" refers to the maximum poresize of the material. The practitioner determines pore size usingconventional bubble point methodology, as described in PharmaceuticalTechnology, May 1983, pages 36 to 42.

As a threshold requirement, the pore size selected must make theboundary 46 impermeable to the vascular structure that forms close tothe boundary 46. Penetration of the pores by the vascular structurebreaches the integrity of the boundary 46, exposing the implanted cellsto the complete immune response of the host. Generally speaking, poresizes less than about 2 microns will block the ingress of vascularstructures.

The ultimate pore size selected also depends upon the species of thehost and the biologic relationship between the host and the donor of theimplant tissue cells.

When the implanted cells are from another animal species (i.e.,xenografts), the pore size must be sufficient to prevent the passage ofboth inflammatory cells and molecular immunogenic factors from the hostinto the implant tissue chamber. As used in this Specification,"molecular immunogenic factors" refers to molecules such as antibodiesand complement.

Pore sizes sufficient to block passage of both inflammatory cells andmolecular immunogenic factors in humans lie in the range of about 0.015micron. Of course, these pore sizes are also impermeable to vascularstructures.

When the implanted cells are from the same animal species but having adifferent genetic make up (i.e., allografts), the pore size usually mustbe sufficient to prevent the passage of only inflammatory cells from thehost into the implant cell chamber. In allografts, molecular immunogenicfactors do not seem to adversely affect the viability of the implantedcells. Still, somedegree of tissue matching may be required for completeprotection.

Pore sizes sufficient to block passage of inflammatory cells in humanslie in the range of below about 0.8 micron. These pore sizes, too, areimpermeable to vascular structures.

When the implanted cells are isografts (autologous implants ofgenetically engineered cells), the pore size must be sufficient only toprevent the isografts from entering the host. Still, with isografts, thepore size selected must also prevent ingress of vascular structures.

Boundary Strength

The boundary 46 has an ultimate strength value that is sufficient towithstand, without rupture, the growth of new vascular structures, thegrowth of new cells within the chamber 18/18', and other physiologicalstresses close to the host tissue. Keeping the boundary 46 secureassures isolation of the implanted cells from both the immunogenicfactors and inflammatory cells of the host.

These physiological stresses are caused when the host moves about incarrying out its normal life functions. The proliferation of implantedcells and the growth of vascular structures 54 also contributes to thephysiological stresses close to the boundary 46. The stresses challengethe physical integrity of the boundary 46 by stretching or otherwisedeforming it.

Absent a sufficient ultimate strength value, normal physiologicalstresses can rupture the boundary 46, exposing the implanted cells tothe full effect of the host's immune and inflammatory systems.

The inventors presently believe that ultimate strength values sufficientto withstand physiological stresses close to the host tissue withoutrupture in animals lie above about 100 pounds per square inch (PSI). Incomparison, the ultimate strength value for PVA hydrogel microcapsulesis only about 2 to 2.5 PSI.

The ultimate strength values are determined by measuring the tensilestrength of the material. Tensile strength is measured by ASTM D-412.

Metabolic Transit Value

The boundary 46 also has a metabolic transit value that sustains a fluxof nutrients into the chamber 18 and waste products from the chamber 18sufficient to sustain the viability of the implanted cells during theischemic period.

The metabolic transit value takes into account the permeability value(P) and the porosity value (PORE) of the boundary 46.

The Permeability Value

The permeability value (P) is the measure of the amount of solute thattravels through the boundary per unit time and unit surface area, givensome fixed external solute concentration (measured in cm/sec in thisspecification). Example 1 sets forth a methodology for determining thepermeability value according to this aspect of the invention.

The Porosity Value

The porosity value (PORE) represents the space in the boundary 46 thatdoes not contain material, or is empty, or is composed of pores.Expressed as a percentage, the porosity value (PORE) measures the %volume of the boundary 46 that is not occupied by boundary material.

To derive the porosity value PORE (in %) for materials having a POREequal to or greater than 10%, the practitioner uses the followingformula:

    PORE=100(1-(ρ.sub.b /ρ.sub.m)

where:

ρ_(b) is the density of the boundary as determined from its weight andvolume, and

ρ_(m) is the density of the boundary material.

To derive the porosity value PORE (in %) for materials having a POREless than 10%, the practitioner uses using a scanning electronmicroscope to obtain the number of pores and their average diameter onthe boundary. PORE is then derived according to the following formula:

    PORE=Nπ(d.sup.2 /4)

where:

N is the pore density and equals (p_(n) /a),

p_(n) is the number of pores in the boundary,

a is the total area of the boundary (in cm²), and

π is the transcendental constant 3.1416 . . . ,

d is the average diameter of the pores (in cm).

The inventors have found that, above a threshold minimum porosity value,the permeability value is the principal influence upon the overallmetabolic transit value. Still, below the threshold minimum porosityvalue, the metabolic transit value must also take into account theporosity value and the physical structure of the porous boundary 46.These considerations will be discussed later in greater detail.

To simplify the selection of a boundary 46, the inventors recommend theuse of boundaries having a porosity value (PORE) greater than theobserved minimum threshold value. Then, metabolic transit value and thepermeability value can be treated as the same.

Referring now to FIG. 23, a further embodiment of the assembly of thepresent invention is illustrated. Generally, the device 100 consists ofa cassette 102 including a cell cavity 104 that is received within apouch-like structure 106.

The cassette 102 in the embodiment illustrated in FIG. 23 preferablyincludes two walls 108 and 110 that provide an immuno-isolating membranebarrier. The immuno-isolating membranes 108 and 110 are provided toprotect cells 112 located within the cell cavity 104 of the cassette 102from the host's immune system. Although two membranes 108 and 110 areillustrated in the preferred embodiment of the cassette 102, it shouldbe appreciated that the cassette can be defined by a single membrane ormultiple membranes.

The assembly 100 includes a second structure, pouch, 106 for receivingthe cassette 102. The pouch 106 provides an outer vascularizing membrane114. This membrane 114 can be substantially similar to the vascularizingmembranes discussed above with respect to other embodiments of theimplant assembly.

As noted above, in an embodiment, the second structure 106 has a pouchshape. The pouch 106 serves to contain the cassette 102 and supportvascularization of the implantation site.

The pouch 106 is defined by the vascularizing membrane 118 that furtherdefines a cavity 115 that is large enough to house the cassette 102. Tothis end, in an embodiment, the pouch 106 is defined by two wall members116 and 118 that are sealed along their edges. To insert the cassette102, an opening 120 is provided at one end of the pouch 106.

Of course, the shape of the pouch 106 is a function of the shape andsize of the cassette 102. A variety of different shapes can be utilizedto define the pouch 106.

The membranes of the cassette and pouch, 108 and 110 and 114,respectively, are constructed so that chemical interaction between thepouch 106 and cassette 102 and surrounding body blood vessels isconducted through each membrane barrier. However, although thevascularizing membrane 114 is capable of supporting blood vessels fromthe host, the body's immune system is prevented from attacking the celltissue inside the cassette 102 by the immuno-isolating membranes 108 and110.

As noted previously, the cassette 102 provides a cell cavity 104 that isfilled with cells 112. In a preferred embodiment, cell spacers 124 and126 are provided to create a suitable cavity 104 capable of tissueprotection. The spacers 124 and 126 serve to maintain optimum celldepth, cell distribution, and/or external shape of the cassette 102.External shape is a function of the surface area required to insure cellto blood vessel interaction.

In use, cells 112 are placed within the cell cavity 104 of the cassette102. When filled with cells 104, the completed cassette 102 is ready forimplantation in the pouch 106. In the alternative, after being loadedwith cells, the cassette 102 is ready for testing or storage in anutrient bath.

In order to securely position the cassette 102 within the pouch 106,sealing means can be located on portions of the cassette 102. Forexample, a layer of sealing gel 130 can be located on portions of themembrane wall 108. The sealing gel will seal the cassette 102 to aninterior wall of the pouch 106.

In use, the pouch 106 can be implanted within the host before insertionof the cassette 102 into the pouch. This will allow proper placement ofthe pouch 106 within the host. Additionally, this can insure that thepouch 106 is properly vascularized and accepted prior to insertion ofthe cassette 102.

When the pouch 106 is prevascularized prior to insertion of the cassette102, the cavity or interior of the pouch is entirely enclosed, i.e., thepouch does not include an opening. The cassette 102 can be inserted inthe pouch 106 by opening the pouch. This can be done by separating thewalls of the pouch 106 at an end thereof, severing a portion of thepouch, or other means.

Cassette 102 placement or retrieval is possible through the pouch 106opening or closure. The pouch 106 opening or closure can be located in avariety of locations on the pouch. Likewise, the means for opening orclosing the pouch 106 can comprise a variety of techniques. Such meansinclude heat seal, sonic welding, clips, and other methods.

The present invention offers the distinct advantage of retrieval withoutcomplete disruption of the membrane vascularization. Follow up tissuetesting from the retrieved cassette 102 may indicate functionality.Refilling the pouch 106 with the new cassette 102 can be utilizedpursuant to the present invention.

The closure means, for closing the pouch 106 opening 120, afterplacement of the cassette 102 can be constructed so as to provide thesurgeon with easy location of the device 106. In this regard, clips canprovide a reference indicator for internal and external device location.Non-evasive external device location offers distinct advantages oversurgical alternatives. Location indicators can also be utilized forpositioning other devices such as needles, biopsy probes, futuredevices, and the like near the implanted device.

Of course, a variety of other methods can be used to reseal the accessopening. These other methods include ultrasonic welding, solventbonding, etc.

Due to the resealable access opening 120 of the pouch 106, not only isan easy method of loading cells in the assembly provided, but theassembly can be recharged by placing new cells within the assembly aftera predetermined time. To this end, after the assembly has beenimplanted, when necessary, a surgeon can create an incision in the skinexposing an end portion of the assembly. The surgeon can then access theinterior 115 of the assembly by cutting open a portion of the pouch 106.

Additionally, as noted above, the assembly allows one to implant thepouch 106 without cells for prevascularization of the assembly.Prevascularization of an assembly and the benefits inherent therein isdisclosed in U.S. patent application Ser. No. 08/180,018, the disclosureof which is hereby incorporated by reference.

As the following Example 1 shows, the inventors have discovered thatthere is a direct correlation between the metabolic transit value andimplanted cell survival during the ischemic period.

EXAMPLE 1

Embryonic lungs enclosed in membrane chambers having differentpermeability values were implanted in subcutaneous sites in rats.

1. Permeability

The permeability values for the membrane chambers were obtained forinsulin diffusion in a conventional benchtop diffusion chamber, made byCrown Glass Company, Somerville, N.J. (Part Number DC-100), usingradioactively labeled (¹²⁵ I) insulin as the solute (obtained from ICNBiochemicals). The diffusion chamber had two chambers (which will becalled Chambers A and B), each with a volume of 3 ml. The diffusionchamber presented a membrane surface area between the two chambers(where diffusion occurs) of 0.7 cm².

The practitioner cuts the membrane material to be tested to apredetermined, known size.

If the membrane is hydrophobic, the practitioner wets the membranebefore conducting the permeability test, using conventional wettingtechniques.

The practitioner places the membrane in the diffusion chamber. Theassembly of the diffusion chamber locates the membrane between the twochambers of equal volume, called Chamber A and Chamber B. In this way,the practitioner also fixes the cross sectional area (A) of themembrane. The diffusion chamber is uniformly heated to a temperature ofabout 37 degrees C. during the test.

The practitioner loads equal amounts of buffer solution into Chamber Aand Chamber B. The buffer solution can vary. In this Example, thepractitioner can use phosphate buffered saline, 0.5% BSA as the buffersolution.

The practitioner then loads equal amounts of unlabeled (non-radioactive)insulin (about 3.4 micro units/ml) into Chamber A and Chamber B. Porcinepancreas insulin purchased from Sigma with an activity of 26.1 units/ml,or comparable material, can be used. The unlabeled insulin occupies anyadsorption sites that may be present.

The practitioner uniformly stirs the fluids within the chamber at about600 RPM, using a magnetic stir plate and magnetic stir rods (about 1 cmin length) placed in each Chamber A and B. The practitioner allows thesystem to equilibrate for about one hour.

The practitioner then removes a selected volume of buffer solution fromChamber A and adds back an equal volume of radioactive insulin. Theradioactive insulin suspension is filtered before use to remove free ¹²⁵I.

While stirring the fluids within Chamber A and Chamber B, thepractitioner draws equal aliquots of fluid from each Chamber A and B(e.g. about 15 uL) at 2, 4, 6, 8, 10, 15, and 30 minute intervals.

The practitioner then counts the radioactivity levels in the samplesusing a gamma counter.

The practitioner determines the change in the counts (i.e., insulinconcentration) in Chambers A and B per unit of time, suitably correctedfor background noise.

The practitioner graphs the count and time pairs for each Chamber interms of time versus the counts (with the counts being the Y-coordinatesand time being the X-coordinates), restricting the analysis to pointsfor which the counts in Chamber B are less than about 10% of the initialcounts in Chamber A. The practitioner then derives a linear equation,fitting the range of counts (y) over the set of times (x) for eachChamber according to the following equations:

For Chamber A:

    Y.sub.a =Y.sub.intercept -(N.sub.a *X)

where

Y_(intercept) is the count value where the graph intersects the Y axis,and

N_(a) is the slope of the Chamber A graph.

For Chamber B:

    Y.sub.b =Y.sub.intercept +(N.sub.b *X)

where

Y_(intercept) is the count value where the graph intersects the Y axis,and

N_(b) is the slope of the Chamber B graph.

The practitioner preferably uses a commercially available computerprogram to simplify the derivation process described above.

The practitioner then derives the permeability value (P) according tothe general expression: ##EQU1## where V_(b) is the volume of Chamber B

dM_(b) /dT is the change in counts in Chamber B per unit time, which isthe slope of the B graph derived above (N_(b)),

P is the permeability value,

A is the area of the boundary tested, and

M_(a) -M_(b) is the mass gradient of insulin across the membrane.

The practitioner knows V_(b) and A, which remain constant throughout thetest. The practitioner also knows dM_(b) /dT, the slope of the graph forChamber B (N_(b)) from the linear equation derived for Chamber B. Thepractitioner converts the units of N_(b) (counts per min/min) intocounts per minute/sec by dividing by 60 (the number of seconds in aminute).

The practitioner calculates M_(a) by solving the linear equation derivedfor Chamber A for y when t=15 minutes (i.e., the mid point time for thetest). By using the mid point time for the test, the practitionerobtains an average value for the period of the test. The practitionersimilarly calculates M_(b) by solving the first order linear equationderived for Chamber B for y when t=15 minutes. From these values, thepractitioner calculates M_(a) -M_(b).

The practitioner can now derive the permeability value (in cm/sec) asfollows: ##EQU2##

Actually, the permeability value derived also includes the boundarylayer effects that are associated with inevitable stagnate fluid layersat the membrane surface in Chambers A and B during the test. To arriveat the "true" intrinsic permeability value for the boundary, thepractitioner would have to adjust for the boundary layer effects.However, for the purposes of this invention, a knowledge of the inherentmembrane permeability is not essential, because it will be proportionalto the experimental permeability value determined following themethodology detailed above.

Yet, the practitioner can follow the foregoing methodology to quantifythe relative permeability values for selected boundaries, since boundarylayer effects will remain constant as long as the stirring method usedremains the same.

The disclosed methodology can be used to assess whether a given boundaryfits the criteria established for the permeability value according tothis aspect of the invention.

2. Porosity

The porosity values (PORE) of the boundaries tested ranged from lessthan about 15% to greater than about 70%.

3. Determining Cell Survival

Embryonic lungs were removed from Lewis rat embryos between days 13.5and 17.5 of development. The lungs were kept on ice in Dulbecco'sModified Eagle's Medium (DMEM), 20% fetal bovine serum. The lungs wereminced until they were approximately 1 mm². Minced lung tissue (5-10 μl)was placed into implant assemblies like those shown in FIGS. 1 to 4. Thelung tissue was encapsulated within test membranes having variouspermeabilities, porosities, and pore sizes. The implant assemblies wereplaced in DMEM (20% fetal bovine serum) at 37 degrees C. until surgery,which occurred within 2 hours. The implant assemblies were implanted insubcutaneous or epididymal fat sites in male Lewis rats for 3 weeks.

After three weeks of implantation, the assemblies were explanted,trimmed of excess fat, and fixed with 2% glutaraldehyde in Sorensen'sbuffer. Sections of the assemblies were stained with hematoxylin andeosin.

Cell survival was scored based upon histological appearance of theimplanted cells. Tissues were scored as "excellent" if they had normalcharacteristics of lung tissue, such as epithelial tubules, cilia, andformed cartilage. Tissues were scored as "good" if the tissue were stillalive, but not well differentiated (for example, a high number ofmesenchymal cells). The tissues were scored as "poor" if no or few cellsremained alive.

In other histology studies using implanted pancreatic cells, survivalassessment would involve analyzing the differentiated function of thepancreatic cells in terms of their insulin release in the response to aglucose challenge.

Table 1 shows the permeability value for those boundaries having aporosity value (PORE) greater than 70%, correlated with the survival ofthe implanted lung tissues.

                  TABLE 1                                                         ______________________________________                                        Membranes with PORE > 15%                                                                   Pore Size or                                                    Membrane      MW Cutoff Permeability                                                                            Tissue Survival                             ______________________________________                                        cellulose acetate.sup.1                                                                     unknown   9         excellent                                   cellulose acetate.sup.1                                                                     unknown   5.3       excellent                                   Biopore ™.sup.2                                                                          0.45 μm                                                                              2.6       excellent                                   polyvinyl difluoride.sup.1                                                                  unknown   2.5       good                                        cellulose mixed ester.sup.2                                                                 1.2 μm 2.0       poor                                        polyvinyl difluoride.sup.1                                                                  unknown   1.7       good                                        polypropylene.sup.3                                                                         0.075 μm                                                                             1.4       poor                                        cellulose acetate.sup.1                                                                     unknown   1.3       poor                                        cellulose mixed ester.sup.2                                                                 0.45 μm                                                                              0.9       poor                                        polyethylene.sup.3                                                                          0.08 μm                                                                              0.9       poor                                        cellulose.sup.4                                                                             300 kD    0.6       poor                                        cellulose.sup.4                                                                             50 kD     0.2       poor                                        ______________________________________                                         *X 10.sup.-4 cm/s                                                             .sup.1 Baxter Healthcare Corporation (Deerfield, Il)                          .sup.2 Millipore Corporation (Bedford, Ma)                                    .sup.3 Hoechst Celanese (Charlotte, NC)                                       .sup.4 Spectrum Medical Instruments (Los Angeles, Ca)                    

Table 2 shows the permeability value of those boundaries having aporosity value (PORE) less than 15%, correlated with the survival of theimplanted cells.

                  TABLE 2                                                         ______________________________________                                        Membranes with PORE < 15%                                                                             Perme-  Tissue                                        Membrane* Pore Size     ability¥                                                                          survival                                      ______________________________________                                        Nuclepore.sup.1                                                                         0.8           4.4     Fair                                          Nuclepore 0.4           3.1     Poor                                          Nuclepore 0.22          2.3     Poor                                          Poretics.sup.2                                                                          0.1           2.2     Poor                                          Poretics  0.08          0.5     Poor                                          Poretics  0.05          1.2     Poor                                          Poretics  0.03          0.9     Poor                                          Poretics  0.01          0.2     Poor                                          ______________________________________                                         *polycarbonate                                                                ¥ X 10.sup.-4 cm/s                                                        .sup.(1) Nuclepore Corporation (Pleasanton, Ca)                               .sup.(2) Poretic Corporation (Livermore, Ca)                             

Tables 1 and 2 demonstrate the direct relationship between the metabolictransit value of the boundary and implanted cell survival. Moreparticularly, the Tables show that implanted cell survival significantlyimproves when the permeability value of the boundary increases.

For the type of cells studied in Example 1, boundaries having apermeability value for insulin less than about 1.5×10⁻⁴ cm/sec, asdetermined using the described methodology, consistently did not supportcell survival, regardless of the porosity value. Yet, boundaries havinga permeability value for insulin greater than about 1.5×10⁻⁴ cm/sec anda porosity value greater than about 15% uniformly supported vigorouscell survival.

Boundaries having a lower porosity value (less than about 15%) alsosupported cell survival (see Table 2). Still, the metabolic transitvalue for these less porous boundaries requires a higher relativepermeability value. For the type of cells studied in Example 1,boundaries having a lower porosity value (less than about 15%) supportedcell survival when the permeability value for insulin was greater thanabout 4.0×10⁻⁴ cm/sec.

The inventors believe that, when considering less porous boundaries,their specific physical structure must also be taken into account. Theless porous interfaces used in Example 1 were track-etched membranes.These membranes have uniform cylindrical pores separated by relativelylarge, nonporous regions.

The poor tissue survival using the low porosity boundaries could be dueto uneven localization of areas of high permeability, or due toconstraints produced by cells on the particular physical properties ofthe track-etched membranes. For example, the cells may be more efficientat plugging up the cylindrical pores of the track-etched membraneseither with cell extensions or cell secretions. Thus, although thetrack-etched membranes have high permeability values in vitro, theresponse of the cells in vivo may prevent the attainment of sufficientmetabolic transit to support the graft cells.

Example 1 demonstrates a methodology that can be followed to identifyfor other cell types the applicable metabolic transit value that assurescell survival during the ischemic period after implantation.

The absolute permeability and porosity values that constitute a givenmetabolic transport value will depend upon the type of cell and themethodologies of determining permeability and porosity. Differentconditions will give different absolute values. Still, regardless of thetest conditions, the relative differences in permeability and porosityvalues derived under constant, stated conditions will serve as anindicator of the relative capabilities of the boundaries to supportimplanted cell viability.

Tables 1 and 2 also show that good tissue survival occurs even withmembrane materials that are subject to the formation of an avascularfibrotic response (the so-called "foreign body capsule"). The fact thatthese membrane materials create this response has, in the past, led tothe widely held view that the formation of the foreign body capsulecaused poor diffusion of nutrients. Example 1 shows the error of thisconventional wisdom.

As Table 1 shows, the use of relative thicker cellulose acetatemembranes with 0.45 micron pore size (130 microns thick) having aninsulin permeability of 0.9×10⁻⁴ cm/sec results in poor tissue survival.On the other hand, the use of relatively thinner cellulose acetatemembranes with the same approximate pore size (10 microns thick) andhaving a greater permeability of 5.3×10⁻⁴ cm/sec results in excellenttissue survival.

The thickness of the membrane does not alter the foreign body response;a foreign body capsule will form whether the membrane is relativelythick or thin. However, membrane thickness does alter the permeabilityvalue.

Thus, the cells died when the thicker boundary was used, not because ofthe formation of the foreign body capsule, but because of poor nutritionand poor waste removal due to the low permeability of the thickerboundary. The tissue survived when the thinner boundary is used, becausethe higher permeability provided improved cell nutrition and improvedwaste removal to support cell metabolism, even when the same foreignbody capsule forms.

EXAMPLE 2

In an experiment, the practitioner grew RAT-2 fibroblasts (ATCC CRL1764) in 20% Fetal Bovine Serum, 2 mM 1-glutamine, and DMEM (Sigma)(high glucose) until 100% confluent. The RAT-2 cells were split 1:2 inthe above media, 16 to 24 hours before surgery.

On the day of surgery, the cells were washed with 15 ml of HBSS (noions) and trypsinized off the culture flask. The practitionerneutralized the trypsin by adding 5 ml of the above media. Thepractitioner pelleted the cells by centrifugation (1000 rpm, 10 minutes,at 22 degrees C.).

The pelleted cells were counted and resuspended in media in threeconcentrations: 5.3×10³ cells/10 μl; 5.8×10⁵ cells/10 μl; and 5.8×10⁶cells/10 μl.

Implant assemblies like that shown in FIGS. 1 to 4 having boundaries ofdiffering permeability values were made. The permeability values rangedfrom 0.2×10⁻⁴ cm/sec to 9×10⁻⁴ cm/sec (see Tables 1 and 2 to follow).The total boundary area for each assembly was about 0.77 cm².

The various cell concentrations were loaded into the assemblies. Thepractitioner implanted the assemblies both subcutaneously and within theepididymal fatpad of host rats.

After 3 weeks, the assemblies were explanted and examinedhistologically, as described previously.

The inventors observed that assemblies loaded with 5.8×10³ cells and5.8×10⁵ cells displayed excellent results, given sufficient boundarypermeability values. After 3 weeks of implantation, the initial load of5.8×10⁵ cells proliferated to approximately 2.0×10⁷ cells. The inventorsobserved that assemblies having higher initial loads of 5.8×10⁶ cellsdisplayed poorer results.

Lower initial loads (less than 5×10⁶) were able to survive the ischemicperiod and even proliferate 30 to 3000 fold. The final cell counts inthe assemblies with lower initial loads were three times higher than theinitial load of the assemblies that failed because of higher initialloads. Thus, high loads of cells (greater than 5×10⁶) are unable tosurvive during the ischemic period, yet the same cell loads are able tosurvive after the ischemic period as progeny of the cells from lowerinitial loads.

Close Vascularization at the Boundary (1) Presence of AngiogenicMaterial

Neovascularization close to the boundary is essential to the long ternsurvival of the implanted cells within the host. The inventors havefound that the host will not grow new vascular structures 54 close tothe boundary (as FIGS. 24 and 25 show), unless it is stimulated to doso. Without proper stimulation, the ischemic period never ends, becausea classical foreign body reaction occurs.

The assembly 10 therefore includes an angiogenic material 56 forstimulating neovascularization close to the boundary.

The specific identity of the angiogenic material 56 is not known. Still,the inventors have determined that the presence of certain cellsstimulate neovascularization, while others do not.

For example, the presence of lung tissues; pancreatic islets; adultpancreatic ducts; and cultured cell lines of fibroblasts, mammary gland,and smooth muscle cells induces or stimulates neovascularization, whencompared to the vascularization on control grafts where these cell typeswere not present.

On the other hand, the presence of primary skin fibroblasts andmicrovascular endothelial cells do not induce neovascularization.

The inventors believe that certain cells induce or stimulateneovascularization by secreting angiogenic factors. Because the stimuluscrosses membranes that are impermeable to cells, it must be a molecularsignal that the living cell generates. This further underscores the needto support the implanted cells during the ischemic period. If angiogenicsource cells perish, the molecular signal stops, and theneovascularization process comes to a halt.

According to this aspect of the invention, when cells are implanted thathave a desired therapeutic effect, but do not secrete angiogenicmaterial, the assembly 10 includes a separate angiogenic source cell ormaterial 56.

Following the invention, the practitioner selects an boundary 46 havinga sufficient metabolic transit value to support the viability of theimplanted cells, i.e., the angiogenic source cells and othernon-angiogenic, therapeutic cells (when present) implanted with them.The practitioner also selects a pore size and ultimate physical strengthto make the boundary 46 impermeable to the neovascular growth that theangiogenic source cells stimulate.

Alternatively, the practitioner may coat the exterior of the boundary 46itself with an angiogenic material 56. Of course, the coated boundary 46still must have sufficient pore size, ultimate strength, and metabolictransit value to sustain the cells 12 isolated behind the boundary 46.

Because the new vascular structures 54 cannot penetrate the boundary 46,and because the angiogenic signal to the host continues, the newvasculature proliferates close to the boundary 46.

As FIG. 21 shows, when the cells 12 die during the ischemic period, andclose vascularization is not stimulated, the fibroblasts of the foreignbody capsule 52 become closely packed and dense. However, as FIG. 20shows, when the cells 12 survive the ischemic period, and the process ofclose vascularization is stimulated, the fibroblasts of the foreign bodycapsule 52 is altered to form a less dense and more dispersed structure.

(2) Conformation for Close Vascularization

In the preferred embodiment, the porous boundary 46 includes aninterface 47 with the host tissue that is characterized by a structuralconformation that further enhances the growth of vascular structures bythe host close to the boundary.

To achieve this result, each wall element 32/32' and 34/34' of theassemblies 10/10' includes a first porous region 58 and a differentsecond porous region 60. The first porous region 58 comprises theboundary 46 previously described. The second porous region 60 comprisesthe interface 47.

The first porous region 58 faces the implanted cells 12 (see FIG. 20).The first porous region 58 has the boundary characteristics, abovedescribed, of pore size; ultimate physical strength; and metabolictransit value. It is this region 58 that isolates the implanted cellsfrom the immune mechanisms of the host, while sustaining their viabilitythrough the flux of nutrients and wastes during the ischemic period.

The second porous region 60 faces the host tissue 44 and forms theinterface 47 with it (see FIG. 20). The second porous region 60 has anarchitecture that enhances the formation of vascular structures 54 closeto the boundary 46. The formation of these vascular structures 54 withinthe second region 60 mark the end of the ischemic period.Vascularization in the second region 60 sustains the viability of theimplanted cells 12 after the ischemic period ends.

A foreign body capsule 52 still forms about the implanted assembly 10.However, close vascularization within the second porous region 60 canalter the normal configuration of the foreign body capsule 52. As FIG.20 shows, a life sustaining vascular bed forms within the capsule 52close to the boundary 46, keeping flattened macrophages, foreign bodygiant cells, and fibroblasts from pressing against and blocking theboundary 46.

Because of the pore size, strength, and permeability characteristics ofthe porous first region 58, it is impermeable to the neovasculature 54formed in the second region 60.

The inventors believe that close vascularization occurs if the threedimensional conformation of second region 60 creates certain hostinflammatory cell behavior.

The inventors have observed by light and electron microscopy that closevascularization occurs if, in the initial period of implantation, atleast some macrophages entering the material are not activated.Activated macrophage are characterized by cell flattening.

The inventors observe close vascularization in regions of an implantwhere the macrophages that have entered the cavities of the materialretain a rounded appearance when viewed through light microscopy(˜400×). At 3000× (TEM) the rounded macrophage is observed to havesubstantially conformed to the contours of the material. Although thereis a correlation with macrophage shape, it is not clear that macrophagescontrol the observed, response. However, it is clear that invasion ofthe structure by host cells is required. Although the bulk of the cellsappear to be macrophages, it is possible that other inflammatory cellscontrol the response, therefore the inventors refer to the invadingcells as "inflammatory cells," which include but are not limited tomacrophages.

On the other hand, foreign body capsule formation occurs when, in theinitial period of implantation, inflammatory cells in contact with theimplant material flatten against those portions of the material whichpresent an area amenable to such flattening behavior by an inflammatorycell.

The material for the second region 60 that results in formation of closevascular structures is a polymer membrane having an average nominal poresize of approximately 0.6 to about 20 μm, using conventional methods fordetermination of pore size in the trade. Preferably, at leastapproximately 50% of the pores of the membrane have an average size ofapproximately 0.6 to about 20 μm.

The structural elements which provide the three dimensional conformationmay include fibers, strands, globules, cones or rods of amorphous oruniform geometry which are smooth or rough. These elements, referred togenerally as "strands," have in general one dimension larger than theother two and the smaller dimensions do not exceed five microns.

In one arrangement, the material consists of strands that define"apertures" formed by a frame of the interconnected strands. Theapertures have an average size of no more than about 20 Am in any butthe longest dimension. The apertures of the material form a framework ofinterconnected apertures, defining "cavities" that are no greater thanan average of about 20 pn in any but the longest dimension.

In this arrangement, the material for the second region has at leastsome apertures having a sufficient size to allow at least some vascularstructures to be created within the cavities. At least some of theseapertures, while allowing vascular structures to form within thecavities, prevent connective tissue from forming therein because of sizerestrictions.

Further details of the material are set forth in copending U.S.application Ser. No. 735,401 entitled "Close Vascularization ImplantMaterial" filed Jul. 24, 1991, which is incorporated into thisSpecification by reference.

Making a Boundary

FIGS. 11 to 16 show a method of making a preferred embodiment of thewall elements 32 and 34 that forms the boundary. The method integrallyjoins material selected for the first region 58 to another materialselected for the second region 60. The two joined materials form thecomposite, or laminated, structure 72 shared by both wall elements 32and 34. The laminated structure 72 joins the interface 47 to theboundary 46.

In the illustrated embodiment, a porous PTFE membrane material having athickness of about 35 microns and a pore size of about 0.4 micron isselected for the first region 58. This material is commerciallyavailable from Millipore Corporation under the tradename Biopore™.

The porous material selected for the first region 58 has a thickness ofabout 30 microns and an ultimate (tensile) strength value of at least3700 PSI, which is well above the desired minimum value. The selectedmaterial has pore size of 0.35 microns, which blocks the passage ofinflammatory cells. The selected material has a permeability value forinsulin of 2.6×10⁻⁴ cm/sec and a porosity value of greater than 70%. Themembrane therefore meets the metabolic transit value requirements.

It should be appreciated that other, comparable materials can meet thestated requirements for the first region 58. For example, polyethylene,polypropylene, cellulose acetate, cellulose nitrate, polycarbonate,polyester, nylon, and polysulfone materials can be used. Mixed esters ofcellulose, polyvinylidene, difluoride, silicone, and polyacrylonitrilecan also be used.

In the illustrated embodiment, a membrane material made by W. L. Goreand Associates (Elkton, Md.) under the tradename Gore-Tex™ is selectedfor the second region 60. The Gore-Tex™ material comprises a microporousmembrane made from PTFE. The membrane is 15 microns thick and has a poresize of 5 microns. Polyester strands 61 join the PTFE membrane to form abacking for it. The backing has a depth of about 120 microns.

The Gore-Tex™ material also has an ultimate strength value well abovethe desired minimum value. The conformation of the polyester strands 61also meets the criteria, set forth earlier, for promoting the growth ofneovascular structures.

In Step 1 (see FIGS. 10 and 11), the practitioner secures the edges of astrip of the Gore-Tex™ material (second region 60) to a lamination slide62, with the polyester backing 61 facing the slide 62.

In Step 2 (see FIG. 13), the practitioner places 2 or 3 laminationslides 62 side-by-side on a work surface. Using a syringe 64, thepractitioner applies cement or adhesive in continuous filaments 66 in aback and forth pattern across the lamination slides 62. The practitionertouches the syringe tip 64 to the work surface at the end of eachfilament 66 to begin a new filament 66.

Step 2 forms a criss-crossing pattern of cement filaments 66 across thesecured strips of the second region material, as FIG. 13 shows.

The cement selected can vary. For example, the cement can be celluloseacetate or similar epoxy material. In the illustrated embodiment, thecement comprises a mixture of Vynathene EY 90500 EVA resin and toluene(made by Mallinckrodt).

In forming the EVA cement mixture, the practitioner adds about 30 gramsof resin and an equal amount of toluene to a bottle. The practitionerseals the bottle to allow the resin to dissolve. The bottle may beperiodically shaken to accelerate this process.

The relative amounts of resin and toluene may have to be slightlyadjusted to arrive at the right consistency for the cement. If thecement is too thin to form continuous filaments when applied, use lesstoluene. If the cement is to viscous to be expressed from the syringe,use more toluene. Small changes in the amount of toluene added result issignificant changes in the viscosity of the cement.

In Step 3 (as FIG. 14 shows), the practitioner places preformed stripsof the Biopore™ membrane material (first region 58) upon the cementfilaments 66 applied in Step 2. In the illustrated embodiment, thepractitioner precuts the Biopore™ membrane material into disks havingthe diameter desired for the wall elements 32 and 34.

In Step 4 (as FIG. 15 shows), the practitioner lays a strip of releasematerial 68 (like Patapar) over the first region material 58 and coversthe layered structure with another lamination slide 70. The practitionerclamps the lamination slides 62 and 70 together, bringing the membranelayers into intimate contact.

In Step 5, the practitioner places the clamped lamination slides 62 and70 in an oven for about 5 to 10 minutes at a temperature of about 80degrees C. The heat melts the EVA cement.

In Step 6, the heated lamination slides 62 and 70 are allowed to cool toroom temperature. Upon cooling and solidification, the filaments 66securely join the Biopore™ membrane material to the Gore-Tex™ membranematerial. The practitioner then unclamps the lamination slides 62 and 70and removes the finished composite structure 72 (in strips).

In Step 7 (as FIG. 16 shows), the practitioner lays the compositestructure 72 strips on a polypropylene cutting slab 74. The practitioneraligns a presized punch 76 over each precut disk, striking the punchwith a hammer. The practitioner thereby frees the wall elements 32 or 34formed of the composite structure of the desired dimensions. Smallscissors may be used to snip any adherent polyester strands not cut bythe die.

Implant assemblies 10/10' are made using the wall elements in the mannerpreviously described.

It should be appreciated that the first region material 58 can beapplied to the second region material 60 by various alternative means toform the laminated structure 72. For example, the first region material58 can be extruded in place upon the second region material 60.

EXAMPLE 3

Assemblies like that shown in FIGS. 1 to 4 and constructed according tothe foregoing process have been successfully used to accomplish completecorrection of diabetes in partially pancreatectomized andstreptozotocin-treated rat hosts. The animals were corrected up to 293days. Upon removal of the implants, the animals reverted to a diabeticstate. Histology of the implants revealed the presence of vascularstructures close to the boundary.

These assemblies presented a boundary area of about 0.77 cm². Eachassembly sustained an initial cell load of about 600 pancreatic islets(or about 600,000 pancreatic cells).

When implanted, the assemblies sustained cell densities of about 200,000islets/cm³. These assemblies, made and used in accordance with theinvention, supported 8 times more pancreatic islets in a given volumethan the CytoTherapeutics assemblies (having cell densities of only25,000 islets/cm³).

Deriving a Therapeutic Loading Factor

As earlier described, one aspect of the invention provides the abilityto identify a metabolic transit value associated with a given cell type.Knowing the required metabolic transit value, in turn, makes it possibleto identify the clinically practical region of operation, where compactimplant assemblies can sustain therapeutically large volumes of cells.

This aspect of the invention provides the methodology to derive and usea therapeutic loading factor (L) to characterize and predict theclinical effectiveness of a given implant assembly for a given celltype.

The therapeutic loading factor (L) takes into account the number ofcells (N) that are required to be implanted to achieve the desiredtherapeutic effect; the effective area (A) of the boundary between theimplanted cells and host that the host can be reasonably expected totolerate; and the metabolic transit value (T) needed to sustain cellviability.

The therapeutic loading factor for a given implant assembly and givenimplanted cell type can be expressed as follows:

    L.sub.c =(A/N.sub.c)*T.sub.min

where

c is the given cell type,

L_(c) is the therapeutic loading factor for the given cell type,

A is the area of boundary between the implanted cells and the hostoffered by the given implant assembly,

N_(c) is the number of cells supported by the boundary area (A), and

T_(min) is the minimum metabolic transit value that will support cellsurvival during the ischemic period, determined according themethodology set forth in Example 1.

If the practitioner selects boundaries having a porosity value ofgreater than 15%, then the permeability value (P) alone can be used toexpress the metabolic transit value (T). The therapeutic load factor canthen be expressed:

    L.sub.c =(A/N.sub.c)*P.sub.min

where P_(min) is the minimum permeability value that will support cellsurvival during the ischemic period.

In the assemblies described in Example 3, the observed ratio between theboundary area and the number of implanted cells (A/N_(c)) for thesuccessful implantation of pancreatic cells was 128 μm² /pancreaticcell. The inventors believe that a somewhat larger ratio of about 150μm² /pancreatic cell will provide a satisfactory margin for varianceamong different hosts.

As earlier discussed, given a boundary porosity value of greater than15%, a permeability value (P) greater than about 1.5×10⁻⁴ cm/sec forinsulin should be provided a metabolic transit value that will sustaincell survival during the ischemic period and afterward.

FIG. 22 shows the therapeutic loading curve for pancreatic cellsgenerated based upon the above considerations. The curve displays thepredicted region of cell survival in terms of the boundary area-to-cellnumber ratio A/N (x-coordinate) and permeability value P (y-coordinate)(given a porosity value of greater than about 15%).

FIG. 22 predicts that assemblies operating to the right of thetherapeutic loading curve will sustain implanted pancreatic cells. FIG.22 predicts that assemblies operating to the left of the therapeuticloading curve will not.

The inventors believe that a human diabetic will require thetransplantation of about 250,000 pancreatic islets (or about 250 millionpancreatic cells) to derive a therapeutic benefit. With this in mind,one can calculate a range of sizes for an implant assembly based uponthe A/N ratio.

The equation for calculating the side dimension (L) in cm of a squareimplant assembly based upon the A/N ratio is as follows: ##EQU3## where:the factor 10⁻⁸ converts micron² to cm².

The equation for calculating the diameter (D) in cm of a round implantassembly based upon the A/N ratio is as follows: ##EQU4## where: thefactor 10⁻⁸ converts micron² to cm².

Table 3 lists a range of L's and D's at different A/N ratios for animplant assembly holding 250,000 pancreatic islets

    ______________________________________                                        A/N    A (cm.sup.2)/side                                                                             L(cm)   D(cm)                                          ______________________________________                                        128    160             12.6    14.3                                           150    188             13.7    15.5                                           200    250             15.8    17.8                                           328    410             20.2    22.8                                           463    579             24.0    24.1                                           ______________________________________                                    

Based upon the foregoing considerations, the inventors believe that A/Nratios less than about 200 μm² /pancreatic cell define the operatingregion of implant assemblies that offer compact, clinically practicalimplant boundary areas. FIG. 22 shows this preferred region.

As FIG. 22 also shows, a practitioner can provide an implant assemblythat combines the benefits of compact size with the ability to sustainthe requisite therapeutical number of cells, by selecting a permeabilityvalue for the boundary that achieves a region of operation to the rightof the therapeutic loading curve. The practitioner also selects theprescribed pore size and ultimate physical strength determined inaccordance with the invention.

FIG. 22 shows that the prior art hollow fiber implant assembly made byCytoTherapeutics (described in the Background section of thisSpecification) falls well outside the preferred region of clinicallypractical operation. This assembly offers an A/N ratio of about 328 μm²/pancreatic cell, about 1.5 times the A/N ratio of the invention.

FIG. 22 also shows a prior art hollow fiber implant assembly made by W.R. Grace and Co. (Lexington, Mass.), as reported by Proc. Natl. Acad.Sci. U.S.A., Vol. 88, pp. 11100-11104 (December 1991). Each hollow fiberhad a length of 2-3 cm, and an inside diameter of 0.177 cm. There were200 to 400 pancreatic islets loaded into each fiber for implantation.Taking an average length of 2.5 cm and an average cell load of 300islets, the associated A/N ratio is 463, more than twice the A/N ratioof the invention.

The foregoing establishes a methodology to derive and use a therapeuticloading factor for pancreatic islets. This methodology can be followedto identify a therapeutic loading factor for other cell types and otherranges of metabolic transit values. The absolute value of thetherapeutic loading factor derived will of course depend upon the typeof cell and the methodologies used to determine permeability andporosity. Different conditions will give different absolute values forthe therapeutic loading factor.

Still, regardless of the test conditions, the relative differences inthe A/N ratios, permeability values, and porosity values derived underconstant, stated conditions will serve as a means to characterize andpredict the clinical effectiveness of a given implant assembly for agiven cell type.

In order to characterize the membrane morphology of membranes that canbe used in the embodiments of the present invention, enface images ofmembrane materials were microscopically photographed with a low voltagescanning electron microscope (LVSEM).

Three membrane materials were visually characterized with enface LVSEMviews:

5 μm PTFE membrane, GORE-TEX® L31324 from W. L. Gore and Associates,Inc. is an expanded polytetrafluoroethylene (PTFE) membrane with a poresize controlled to 5 μm. It has a nonwoven spunbound polyester backinglayer as a support. Vender specification of membrane thickness is0.006±0.002 inches.

Reference number: 17222-65-3

Lot number: 41-100593-001

Part number: L31324

0.4 μm PTFEE membrane, BIOPORE® SF1R848E1 from Millipore Corporation isan expanded polytetrafluoroethylene, (PTFE) membrane with a pore sizecontrolled to 0.4 μm. Vender specification of membrane thickness is0.0009±0.0002 inches.

Reference number: 17222-65-2

Lot number: K3JM2594

Part number: SF1R848E1

Woven polyester, SAATIFIL® PES 290/50 from Saati Corporation is a wovenpolyester membrane made of polyester fibers. Vender specification ofmembrane thickness is 0.007 to 0.010 inches.

Reference number 17222-65-1

Lot number: 434180/15/11

Part number: PES 290/50 M PW

Membrane samples for field emission LVSEM analysis were prepared bymounting razor blade cut sections of each membrane on aluminum pin-typeSEM specimen stubs. Avery Spot-O-Glue™ adhesive tabs were used to holdthe samples in place. The mounted specimens were rimmed with silveradhesive paint for improved conductivity and imaging. The mountedspecimens were vacuum sputter coated with palladium to enablereflectance of electrons during SEM observation.

The membrane morphologies are illustrated by FIGS. 24-29 which are asfollows:

FIGS. 24-26 illustrate the first membrane material examined--GORE-TEX®.To this end:

FIG. 24 is a Micrograph at 1000× magnification of the 5 μm Gore-Tex®PTFE membrane surface. Reference number 17222-65-3, Lot number41-100593-001, Part number L31324;

FIG. 25 is a Micrograph at 500× magnification of the 5 μm Gore-Tex® PTFEmembrane surface. Reference number 17222-65-3, Lot number 41-100593-001,Part number L31324; and

FIG. 26 is a Micrograph at 30× magnification of the 5 μm Gore-Tex® PTFEmembrane surface. Reference number 17222-65-3, Lot number 41-100593-001,Part number L31324.

FIGS. 27-28 are micrographs for the second membrane material BIOPORE®.To this end, the Figures are as follows:

FIG. 27 is a Micrograph at 4000× magnification of the 0.4 μm Biopore®PTFE membrane surface. Reference number 17222-65-2, Lot number K3JM2594,Part number SF1R848E1; and

FIG. 28 is a Micrograph at 1000× magnification of the 0.4 μm Biopore®PTFE membrane surface. Reference number 17222-65-2, Lot number K3JM2594,Part number SF1R848E1.

FIG. 29 is a Micrograph at 30× magnification of the Woven SAATIFIL®polyester membrane surface. Reference number 17222-65-1, Lot number434180/15/11, Part number PES 290/50 M PW.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its attendant advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

We claim:
 1. An implant assembly for a host tissue comprising:a firstmember including wall means that define a chamber for holding a secondmember, the wall means including a membrane defining an interface withthe host tissue, the membrane having a conformation that results ingrowth of vascular structures by the host tissue close to the interface;and a second member that is removably received in the first member andincluding a cavity having cells and a membrane having a structure thatisolates the cells within the cavity from the immune response of thehost.
 2. The implant assembly of claim 1 wherein the first memberincludes a resealable opening.
 3. The implant assembly of claim 1wherein the wall means define a chamber that has a substantiallyrectangular cross-sectional shape.
 4. The implant assembly of claim 1including means for resealing an opening of the first member.
 5. Theimplant assembly of claim 4 wherein the means for resealing the openingincludes a removable mechanical means for sealing an opening at an endof the first member.
 6. The implant assembly of claim 1 including aspacer that defines, in part, the cavity for receiving cells.
 7. Theimplant assembly of claim 1 wherein the wall means of the first memberdefines a structure having a substantially pillow-like shape.
 8. Theimplant assembly of claim 1 including means for securing the secondmember in the first member.
 9. An implant assembly for a host tissuecomprising:a pouch including a first wall means defining a chamber forholding a second member, the first wall means including an outervascularizing membrane having a conformation that results in growth ofvascular structures by the host tissue close to an interface between thevascularizing membrane and host tissue; and a second member that can beremovably inserted in the chamber, the second member including a secondwall means defining an interior for receiving cells and defining animmunoisolation barrier that isolates the cells from the immune responseof the host tissue.
 10. The implant assembly of claim 9 wherein thepouch includes a resealable opening.
 11. The implant assembly of claim 9wherein the wall means define a chamber that has a substantiallyrectangular cross-sectional shape.
 12. The implant assembly of claim 9including means for resealing an opening of the pouch.
 13. The implantassembly of claim 12 wherein the means for resealing the openingincludes a removable mechanical means for sealing an opening at an endof the pouch member.
 14. The implant assembly of claim 9 including aspacer that defines, in part, the interior for receiving cells.
 15. Theimplant assembly of claim 9 wherein the walls means of the pouch definesa structure having a substantially pillow-like shape.
 16. The implantassembly of claim 9 including means for securing the second member inthe pouch.